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OPEN
received: 26 October 2016 accepted: 13 February 2017 Published: 20 March 2017
Enterohemorrhagic Escherichia coli pathogenesis: role of Long polar fimbriae in Peyer’s patches interactions Charlotte Cordonnier1,2, Lucie Etienne-Mesmin1,2,†, Jonathan Thévenot1,2,‡, Amandine Rougeron1, Sandra Rénier1, Benoit Chassaing1,†, Arlette Darfeuille-Michaud1,*, Nicolas Barnich1, Stéphanie Blanquet-Diot2 & Valérie Livrelli1,3 Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens whose survival and virulence in the human digestive tract remain unclear owing to paucity of relevant models. EHEC interact with the follicle-associated epithelium of Peyer’s patches of the distal ileum and translocate across the intestinal epithelium via M-cells, but the underlying molecular mechanisms are still unknown. Here, we investigated the involvement of Long polar fimbriae (Lpf) in EHEC pathogenesis. Of the 236 strains tested, a significant association was observed between the presence of lpf operons and pathogenicity. In sophisticated in vitro models of the human gastro-intestinal tract, lpf expression was induced during transit through the simulated stomach and small intestine, but not in the colonic compartment. To investigate the involvement of Lpf in EHEC pathogenesis, lpf isogenic mutants and their relative trans-complemented strains were generated. Translocation across M-cells, interactions with murine ileal biopsies containing Peyer’s patches and the number of hemorrhagic lesions were significantly reduced with the lpf mutants compared to the wild-type strain. Complementation of lpf mutants fully restored the wild-type phenotypes. Our results indicate that (i) EHEC might colonize the terminal ileum at the early stages of infection, (ii) Lpf are an important player in the interactions with Peyer’s patches and M-cells, and could contribute to intestinal colonization. Enterohemorrhagic Escherichia coli (EHEC), as a subgroup of Shiga toxin (Stx)-producing E. coli (STEC), are food borne pathogens responsible for human diseases. In addition to uncomplicated diarrhea, EHEC can cause hemorrhagic colitis (HC) and life-threatening complications such as the hemolytic-uremic syndrome (HUS)1. Ruminants, especially cattle, are a natural reservoir of STEC, and human infection is linked with the consumption of contaminated food. STEC belong to a wide range of serotypes; however, only a limited number has been associated with human disease, among which EHEC O157:H7 is the most prevalent serotype associated with outbreaks and sporadic cases worldwide2. Based on the association of serotypes with diseases of varying severity in humans and with outbreaks or sporadic disease, STEC have been classified into 5 seropathotypes: A (associated with outbreaks and HUS and belonging to the O157:H7 serotype), B (associated with outbreaks and HUS but less commonly than serotype O157:H7), C (associated with sporadic HUS but not with outbreaks), D (associated with diarrhea but not with outbreaks or HUS cases), and E (serotypes that have not been involved in disease in humans)3. To date, the management of EHEC infections only includes supportive therapy, since antibiotics could worsen clinical outcomes4.
1
Université Clermont Auvergne, Inserm U1071, M2iSH “Microbes, Intestin, Inflammation et Susceptibilité de l’Hôte”, USC-INRA 2018, F-63000 Clermont-Ferrand, France. 2Université Clermont Auvergne, MEDIS “Microbiologie Environnement DIgestif Santé”, F-63000 Clermont-Ferrand, France. 3CHU Clermont-Ferrand, Service de Bactériologie, Parasitologie Mycologie, Clermont-Ferrand, F-63000, France. †Present address: Center for Inflammation, Immunity and Infection, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA 30303, USA. ‡Present address: STLO, “Science et Technologie du Lait et de l’Oeuf ” Agrocampus Ouest, INRA, Rennes, 35042, France. *Deceased. Correspondence and requests for materials should be addressed to V.L. (email: [email protected]) Scientific Reports | 7:44655 | DOI: 10.1038/srep44655
1
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Figure 1. lpfOI-141 and lpfOI-154 are associated with the seropathotype A including the most virulent strains. (A,B) The prevalence of lpfOI-141, lpfOI-154 and lpfOI-113 operons was determined by PCR in a total of 236 STEC/ EHEC isolates, and expressed as percentages of the total number of strains according to the strain origin (A) and seropathotype (B).
Survival and colonization of the human gastrointestinal (GI) tract are key features of EHEC infections but remain poorly described due to the lack of relevant models. EHEC viability and expression of virulence genes in the digestive environment have been mostly investigated in oversimplified in vitro models not representative of human physiological conditions5–7. In the human digestive tract, EHEC strains produce Shiga-toxins (Stx) considered to be essential for virulence and major risk factors for severe EHEC infections. Meanwhile, EHEC pathogenesis is not restricted to toxin-mediated effects, and a combination of virulence traits seems to be required, as demonstrated by the intimate bacterial attachment to host epithelial cells leading to the characteristic attaching and effacing (A/E) lesions8,9. The terminal ileum and colon are considered to be the main sites of EHEC colonization in humans 10. In vitro organ culture (IVOC) studies have demonstrated a preferential tropism of EHEC O157:H7 for the Follicle-Associated Epithelium (FAE) of Peyer’s patches, mainly localized in the distal ileum in humans11. It has been hypothesized that the Peyer’s patches-rich distal ileum might represent the initial site of EHEC adhesion and colonization, and EHEC would then spread to other regions of the gut12. Concurrently, the FAE promotes uptake of antigens and microorganisms through specialized epithelial cells with high transcytotic capacity, termed M-cells13,14. A previous study has suggested an interaction of EHEC strains with murine Peyer’s patches, followed by a translocation through M-cells from the gut lumen to underlying tissues. After bacterial uptake by M-cells, Stx induces apoptosis in underlying infected macrophages, which yields to toxin release in the lamina propria. Stx would then enter the bloodstream to reach target organs, leading to severe disease in humans15. However, underlying molecular mechanisms involved in M-cell targeting remain poorly investigated, and the bacterial effectors associated have not yet been defined. Long polar fimbriae (Lpf), first described as putative adhesins in Salmonella enterica serovar Typhimurium, have been shown to attach directly to murine Peyer’s patches16,17. Lpf have also been previously described in Crohn’s disease-associated Adherent-Invasive Escherichia coli (AIEC) as key players in their interactions with M-cells18. Genome analysis of EHEC O157:H7 strain EDL933 revealed the presence of two lpf clusters encoded by O-islands 141 and 154, closely related to Lpf of Salmonella Typhimurium19–21. In EHEC O157:H7, Lpf have been reported to be involved in the adhesion process and micro-colony formation at the surface of cultured cells21,22 probably through binding to extracellular matrix protein23. Recent studies supported the role of Lpf in the induction of host pro-inflammatory responses to EHEC infection24,25. In EHEC O113:H21, a fimbrial cluster related to Lpf has also been identified located at the same position of the O island 154 in EHEC O157:H7 strain EDL93326. LpfOI-113, identified in E. coli strains of other serogroups, may be involved in adherence of E. coli strains to epithelial cells27. In the present study, the roles of Lpf in EHEC pathogenicity and in tropism to Peyer’s patches were investigated using in vitro and in vivo approaches. First, the prevalence of lpf operons was analyzed according to STEC seropathotype and origin, in a collection of 236 strains of known serotype. A strong association of the lpfOI-141 and lpfOI-154 operons and pathogenicity was observed. Then, lpf expression was investigated in relevant dynamic in vitro models of the human digestive tract, with lpf being overexpressed in gastric and small intestinal conditions but not in colonic ones. Using lpf isogenic mutants and their relative trans-complemented strains, we showed that expression of lpf genes is required for an active translocation across M-cell monolayer in vitro and for interactions with Peyer’s patches in mice ileal loops in vivo.
Results
lpfAOI-141 and lpfAOI-154, but not lpfAOI-113, are associated with pathogenic strains. Some STEC
serotypes recovered from animals or food have never been associated with human diseases, therefore STEC strains were classified into seropathotypes, according to association with HUS and outbreaks28. To assess the potential links between lpf operons and STEC pathogenicity, the prevalence of lpfAOI-141, lpfAOI-154 and lpfAOI-113 was investigated in a collection of 236 STEC strains isolated from humans, animals or food and classified according to the source and seropathotype (Fig. 1). Among the 236 strains of known serotype29,30, 175 were isolated from bovine feces, 27 from HUS and HC patients, 24 from food samples and 10 from asymptomatic children. The lpfOI-141 and lpfOI-154 operons were identified in 15 (6.4%) and 30 (12.7%) of the 236 STEC strains, respectively, versus 143 (60.6%) for lpfAOI-113 (Supplemental Fig. 1A and B). However, lpfOI-141 and lpfOI-154 were significantly Scientific Reports | 7:44655 | DOI: 10.1038/srep44655
2
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OPEN
received: 26 October 2016 accepted: 13 February 2017 Published: 20 March 2017
Enterohemorrhagic Escherichia coli pathogenesis: role of Long polar fimbriae in Peyer’s patches interactions Charlotte Cordonnier1,2, Lucie Etienne-Mesmin1,2,†, Jonathan Thévenot1,2,‡, Amandine Rougeron1, Sandra Rénier1, Benoit Chassaing1,†, Arlette Darfeuille-Michaud1,*, Nicolas Barnich1, Stéphanie Blanquet-Diot2 & Valérie Livrelli1,3 Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens whose survival and virulence in the human digestive tract remain unclear owing to paucity of relevant models. EHEC interact with the follicle-associated epithelium of Peyer’s patches of the distal ileum and translocate across the intestinal epithelium via M-cells, but the underlying molecular mechanisms are still unknown. Here, we investigated the involvement of Long polar fimbriae (Lpf) in EHEC pathogenesis. Of the 236 strains tested, a significant association was observed between the presence of lpf operons and pathogenicity. In sophisticated in vitro models of the human gastro-intestinal tract, lpf expression was induced during transit through the simulated stomach and small intestine, but not in the colonic compartment. To investigate the involvement of Lpf in EHEC pathogenesis, lpf isogenic mutants and their relative trans-complemented strains were generated. Translocation across M-cells, interactions with murine ileal biopsies containing Peyer’s patches and the number of hemorrhagic lesions were significantly reduced with the lpf mutants compared to the wild-type strain. Complementation of lpf mutants fully restored the wild-type phenotypes. Our results indicate that (i) EHEC might colonize the terminal ileum at the early stages of infection, (ii) Lpf are an important player in the interactions with Peyer’s patches and M-cells, and could contribute to intestinal colonization. Enterohemorrhagic Escherichia coli (EHEC), as a subgroup of Shiga toxin (Stx)-producing E. coli (STEC), are food borne pathogens responsible for human diseases. In addition to uncomplicated diarrhea, EHEC can cause hemorrhagic colitis (HC) and life-threatening complications such as the hemolytic-uremic syndrome (HUS)1. Ruminants, especially cattle, are a natural reservoir of STEC, and human infection is linked with the consumption of contaminated food. STEC belong to a wide range of serotypes; however, only a limited number has been associated with human disease, among which EHEC O157:H7 is the most prevalent serotype associated with outbreaks and sporadic cases worldwide2. Based on the association of serotypes with diseases of varying severity in humans and with outbreaks or sporadic disease, STEC have been classified into 5 seropathotypes: A (associated with outbreaks and HUS and belonging to the O157:H7 serotype), B (associated with outbreaks and HUS but less commonly than serotype O157:H7), C (associated with sporadic HUS but not with outbreaks), D (associated with diarrhea but not with outbreaks or HUS cases), and E (serotypes that have not been involved in disease in humans)3. To date, the management of EHEC infections only includes supportive therapy, since antibiotics could worsen clinical outcomes4.
1
Université Clermont Auvergne, Inserm U1071, M2iSH “Microbes, Intestin, Inflammation et Susceptibilité de l’Hôte”, USC-INRA 2018, F-63000 Clermont-Ferrand, France. 2Université Clermont Auvergne, MEDIS “Microbiologie Environnement DIgestif Santé”, F-63000 Clermont-Ferrand, France. 3CHU Clermont-Ferrand, Service de Bactériologie, Parasitologie Mycologie, Clermont-Ferrand, F-63000, France. †Present address: Center for Inflammation, Immunity and Infection, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA 30303, USA. ‡Present address: STLO, “Science et Technologie du Lait et de l’Oeuf ” Agrocampus Ouest, INRA, Rennes, 35042, France. *Deceased. Correspondence and requests for materials should be addressed to V.L. (email: [email protected]) Scientific Reports | 7:44655 | DOI: 10.1038/srep44655
1
www.nature.com/scientificreports/
Figure 1. lpfOI-141 and lpfOI-154 are associated with the seropathotype A including the most virulent strains. (A,B) The prevalence of lpfOI-141, lpfOI-154 and lpfOI-113 operons was determined by PCR in a total of 236 STEC/ EHEC isolates, and expressed as percentages of the total number of strains according to the strain origin (A) and seropathotype (B).
Survival and colonization of the human gastrointestinal (GI) tract are key features of EHEC infections but remain poorly described due to the lack of relevant models. EHEC viability and expression of virulence genes in the digestive environment have been mostly investigated in oversimplified in vitro models not representative of human physiological conditions5–7. In the human digestive tract, EHEC strains produce Shiga-toxins (Stx) considered to be essential for virulence and major risk factors for severe EHEC infections. Meanwhile, EHEC pathogenesis is not restricted to toxin-mediated effects, and a combination of virulence traits seems to be required, as demonstrated by the intimate bacterial attachment to host epithelial cells leading to the characteristic attaching and effacing (A/E) lesions8,9. The terminal ileum and colon are considered to be the main sites of EHEC colonization in humans 10. In vitro organ culture (IVOC) studies have demonstrated a preferential tropism of EHEC O157:H7 for the Follicle-Associated Epithelium (FAE) of Peyer’s patches, mainly localized in the distal ileum in humans11. It has been hypothesized that the Peyer’s patches-rich distal ileum might represent the initial site of EHEC adhesion and colonization, and EHEC would then spread to other regions of the gut12. Concurrently, the FAE promotes uptake of antigens and microorganisms through specialized epithelial cells with high transcytotic capacity, termed M-cells13,14. A previous study has suggested an interaction of EHEC strains with murine Peyer’s patches, followed by a translocation through M-cells from the gut lumen to underlying tissues. After bacterial uptake by M-cells, Stx induces apoptosis in underlying infected macrophages, which yields to toxin release in the lamina propria. Stx would then enter the bloodstream to reach target organs, leading to severe disease in humans15. However, underlying molecular mechanisms involved in M-cell targeting remain poorly investigated, and the bacterial effectors associated have not yet been defined. Long polar fimbriae (Lpf), first described as putative adhesins in Salmonella enterica serovar Typhimurium, have been shown to attach directly to murine Peyer’s patches16,17. Lpf have also been previously described in Crohn’s disease-associated Adherent-Invasive Escherichia coli (AIEC) as key players in their interactions with M-cells18. Genome analysis of EHEC O157:H7 strain EDL933 revealed the presence of two lpf clusters encoded by O-islands 141 and 154, closely related to Lpf of Salmonella Typhimurium19–21. In EHEC O157:H7, Lpf have been reported to be involved in the adhesion process and micro-colony formation at the surface of cultured cells21,22 probably through binding to extracellular matrix protein23. Recent studies supported the role of Lpf in the induction of host pro-inflammatory responses to EHEC infection24,25. In EHEC O113:H21, a fimbrial cluster related to Lpf has also been identified located at the same position of the O island 154 in EHEC O157:H7 strain EDL93326. LpfOI-113, identified in E. coli strains of other serogroups, may be involved in adherence of E. coli strains to epithelial cells27. In the present study, the roles of Lpf in EHEC pathogenicity and in tropism to Peyer’s patches were investigated using in vitro and in vivo approaches. First, the prevalence of lpf operons was analyzed according to STEC seropathotype and origin, in a collection of 236 strains of known serotype. A strong association of the lpfOI-141 and lpfOI-154 operons and pathogenicity was observed. Then, lpf expression was investigated in relevant dynamic in vitro models of the human digestive tract, with lpf being overexpressed in gastric and small intestinal conditions but not in colonic ones. Using lpf isogenic mutants and their relative trans-complemented strains, we showed that expression of lpf genes is required for an active translocation across M-cell monolayer in vitro and for interactions with Peyer’s patches in mice ileal loops in vivo.
Results
lpfAOI-141 and lpfAOI-154, but not lpfAOI-113, are associated with pathogenic strains. Some STEC
serotypes recovered from animals or food have never been associated with human diseases, therefore STEC strains were classified into seropathotypes, according to association with HUS and outbreaks28. To assess the potential links between lpf operons and STEC pathogenicity, the prevalence of lpfAOI-141, lpfAOI-154 and lpfAOI-113 was investigated in a collection of 236 STEC strains isolated from humans, animals or food and classified according to the source and seropathotype (Fig. 1). Among the 236 strains of known serotype29,30, 175 were isolated from bovine feces, 27 from HUS and HC patients, 24 from food samples and 10 from asymptomatic children. The lpfOI-141 and lpfOI-154 operons were identified in 15 (6.4%) and 30 (12.7%) of the 236 STEC strains, respectively, versus 143 (60.6%) for lpfAOI-113 (Supplemental Fig. 1A and B). However, lpfOI-141 and lpfOI-154 were significantly Scientific Reports | 7:44655 | DOI: 10.1038/srep44655
2
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